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Molecular microscopy of labeled polynucleotides: Stability of osmium atoms
J. Mol. Biol.
(1977) 117, 387400
Molecular Microscopy of Labeled Polynucleotides : Stability of Osmium Atoms M. D. COLE, J. W. WIGGINS
AND M. BEER
The T. C. Jenkins Departwhent of Biophysics The Johns Hopkins University Baltimore, Md 21218, U.S.A. (Received 20 May 1977, and in revised forwa 25 August
1977)
Polynucleotides and modified polynucleotides reacted with osmium tetroxide and bipyridine or dicarboxybipyridine to bind one or two osmium atoms per nucleotide were viewed in a high-resolution scanning transmission electron microscope under conditions where the osmium atoms are visible. The micrographs show the individual osmium atoms in a configuration determined by their specific chemical bonding with the nucleic acid molecule and with a number and average spacing that is consistent with the expected value. Although the bonds are broken during the imaging, many osmium atoms are found to be contained within the narrow band of the polynucleotide strand and to move less than 5 A between scans of the same area. These results suggest that it is possible to obtain structural information on macromolecules to a resolution of 5 to 10 A using osmium as a specific stain.
1. Introduction Many of the central problems in modern biology can be attacked powerfully if single sites in macromolecules or macromolecular heavy atoms are bound to particular assemblies, and the sites so labeled are recognized in the electron microscope. For example, such specific labeling might be used to localize nucleic acid strands or individual protein molecules in ribosomes, nucleosomes or other nucleoprotein complexes. Considerable chemical information is now available which makes possible the preparation of appropriately labeled macromolecular systems for electron microscopy. The visualization of heavy atoms has now been realized by several laboratories using either the particularly suitable high-resolution scanning transmission electron microscope (Wall et al., 1974; Coleet al., 1976) or conventional electron microscopes (Whiting & Ottensmeyer, 1972 ; Hashimoto et al., 1971; Formanek et al., 1971). The stability of heavy atoms incorporated into macromolecules during the preparation of specimens and during electron microscopy are key questions which have not been adequately answered. Indeed, some notable failures in obtaining structural insight from micrographs of labeled macromolecular systems have cast doubt on the feasibility of the approach (Langmore & Crewe, 1974). In this paper we demonstrate that heavy atoms bound to biological macromolecules are detectable; that over long stretches their number and distribution are consistent with expectation, and that pairs of successive micrographs indicate an average movement of about 3 a for each heavy atom from the first to second micrograph, neglecting those that ha,ve moved so far that they cannot be identified. 3; 387
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2. Materials and Methods Heavy-atom-labeled specimens were prepared as described by Chang et al. (1977) by treating polynucleotides with 5 mM-osmium tetroxide in 5 m&f-bipyridine or 4,4’-dicarboxy2,2’-bipyridine for 24 h at room temperature followed by gel filtration chromatography on Sephadex G50. Polyuridylic acid acquires 1 osmium atom/nucleotide under these conditions. Each thymidine in poly[d(A-T)] will also acquire 1 osmium atom (Chang et aZ., 1977). To obtain a sample containing 2 heavy atoms/nucleotide, polycytidylic acid was allowed to react with o-furfuryl-hydroxylamine in the presence of sodium bisulfite prior to treatment with osmium tetroxide. Under these conditions the modified polymer takes up 2 heavy atoms/nucleotide (Rose & Beer, unpublished work). Specimens prepared in the manner described above contain covalently bound, heavy-atom labels at essentially every nucleotide. 4,4’-Dicarboxy-2,2’-bipyridine was synthesized as described by Case (1946). Thin carbon support films were prepared by indirect evaporation from a resistanceheated rod onto freshly cleaved mica. The thinnest, smoothest films were obtained by relatively slow evaporation. Thickness was assayed in the scanning transmission electron microscope by the electron scattering intensity. A typical film was 30 A thick. The thin films were supported on carbon-coated fenestrated plastic films. A number of bright spots, assumed to be heavy element atoms, are seen in the background of the images. The density of background atoms depends on the preparation techniques and ranges between 2 and 3 atoms/lo3 A”. In an attempt to minimize the number of these spots, spectroscopically pure carbon (National AGKSP) and deionized water (from a Millipore Milli-Q system; resistivity = 18 Mohm cm-‘) were used (Isaacson et al., 1974). Grid materials other than copper, a variety of plastic materials for fenestrated films, and treating the films with chelating agents were tried with no consistent improvement over the level seen in the images presented here. The polynucleotide strands were freed from virtually all contaminating salts by gel filtration chromatography using deionized water as the eluant. This solution was then made 1O-30/o (w/v) benzyldimethylalkylammonium chloride and spread as a monolayer on deionized water (Vollenweider et al., 1975). The polynucleotide preparations were viewed in a high-resolution scanning transmission electron microscope constructed at Johns Hopkins (Wiggins et al., 1974). The beam energy was between 47 and 50 keV in all instances. Total pressure throughout the microscope was below 10mQ Torr. Data were recorded from two electron detectors. Each detector consists of a CaF(Eu) scintillator optically coupled to a photomultiplier. One, designated the central detector, collected the electrons falling within the cone of illumination with a half angle of 13 mrad. The other detector was an annular disc of scintillator collecting the electrons between 13 mrad and approximately 156 mrad. The images shown here consist of the ratio of the annular detector signal to the central detector signal; therefore, they are dark field images. The detector signals and the ratio were recorded by a digital scanning system (Woodruff et al., 1974). The time for which the beam samples each point on the specimen is 30 ps. The electron dosage was determined by two separate methods. One method was a calculation from the known total field emission current, demagnification, and aperture angle. The other method was through calibration of the scintillator-photomultiplier sensitivity. The electron dose in all cases was between lo3 and lo4 e/AZ. The specimen was first searched at low magnification to locate polynucleotide strands. This search resulted in an irradiation of the strand of no more than 10% of the dose in a single high magnification scan. Focusing was accomplished on an adjacent area and then the scan was shifted electrically to the strand where successive scans at high magnification were recorded. The final images were produced by a digital film scanner/writer. Although the ultra-high vacuum environment of the microscope contributes no matter to the sample, contamination can be observed due to surface diffusion of matter (Hart et al., 1970). To avoid contamination, each sample was irradiated by an infra-red lamp while in the airlock (Isaacson et al., 1974). It was then inserted into the high vacuum chamber while still warm. After this procedure there was no detectable contamination. Spatial filtering of the images was accomplished digitally with the PDP 1 l/20 processor,
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which is an integral part of our system. The convolution integral J(Z) = j J(C) *A(.z - [) di was evaluated where I(Z) is the micrograph intensity and A(z) is an envelope function defining the extent of spatial filtering. This integral is identical to .9-1[~(1(z))*9(A(z))], which is the usual form of a spatially filtered function. The A(z) value used W&Ban integer approximation to a 2-dimensional Gaussian function centered on the pixel being evaluated, and including 32, 64 or 128 pixels total. image. The spatial frequencies are attenuated above 0.1 A-l.J(z) is a low frequency The high frequency image K(X) is obtained aa K(z) = I(Z) - J(a) + C, where C, a constant, is chosen large enough that K(z) is always positive. A more conventional Fourier transform and filtering program, or use of a larger memory and more time to perform the convolution described here, would allow a wider choice of the envelope function A(z). The speed of this short filtering r&tine and its availability on the on-line computer make it worthwhile. Comparison of the filtered and unfiltered images presented here and filtering of known test patterns give confidence that no unexpected feature is being introduced into the filtered images. The criteria used to assign a spot aa an osmium atom were that it be more intense than its surrounding in the unfiltered image, that it be more intense than the general background in the filtered image, and that it have a size comparable to the resolution of the microscope. Those spots larger than the microscope resolution and of sufficient intensity M’ere assigned as 2 osmium atoms. Although the assignment technique is quite subjective, ahnost any observer will agree on the quantitative meas&es we have derived from the micrographs within $200/& Figure 6 shows an example of how we have interpreted a port.ion of Fig. l(a).
3. Results Figures 1, 2, 3 and 4 show examples of polynucleotide strands that have been labeled with osmium. The osmium atoms appear as small white spots. In addition, the organic portion of the strand, and probably additional adsorbed organic material, produce diffuse light areas surrounding the osmium atoms. These images show individual atoms in a configuration resulting from their specific chemical association with the nucleic acid molecules. Examination of unreacted polynucleotides shows strands that are only faintly visible, if at all, with no identifiable atoms over those expected from the random background. The intensity of the osmium atoms with respect to the support film, the lack of phase contrast effects in the support film, and the general appearance of the images are all in accord with scanning transmission electron microscope imaging theory (Zeitler 6 Thomson, 1970), calculations of single atom appearance (Engel et al., 1974), and previous scanning transmission microscopy of single atoms (Crewe et al., 1975). If the images are examined closely, linear striations in the direction of the scan lines are seen superimposed on the regular features. These striations indicate movement during the scanning time. The fact that the striations are in the scan direction does not necessarily indicate that the actual movement was in that direction. Movement in any direction in the specimen plane, of period comparable to the time the beam samples each specimen point, will show up as though in the scan direction. The movement may be due to two sources. One possibility is that the components of the specimen move during electron exposure. This seems to be true and is discussed below. Another less fundamental source of movement is from the microscope itself. Instabilities, both mechanical and electrical, may cause abrupt translation of the heam with respect to the specimen. We are currently unable to separate the contribution due to specimen movement from the instrumental instability. The same movements of beam relative to specimen might give rise to a variation in the
intensity
of a single
atom
image
because
the
scattering
power
of an atom
M.
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Fm. 1. Polyuridylic acid labeled with 1 osmium atom/nucleotide using osmium/bipyridino. (a) First high-resolution scan. (b) Second scan taken 8.6 s later. (0) First scan after filtering with the computer to remove low frequency variations in the micrograph. (d) Second scan after filtering. The horizontal width of each micrograph is 410 A.
OSMIUM-
Poly[d( A-T)] labeled ‘(a) First high- resolution
LABELED
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with 1 osmium atom/thymidine using osmium/die acan. (b) Second scan. The horizontal width of each IT
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Fro. 3. Images from Figure
J. W.
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AND
filtering.
M. BEER
(a) First scan. (b) Second scan.
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FIG. 4. Polycytidylic acid labeled with 2 osmium etoms/nucleotide (see Materials and Methods). The specimen was prepared by adsorbing the osmium-labeled polynucleotides to a thin 30 A carbon film and then evaporating a 100 A layer of carbon over the strands. All images have been low-frequency filtered. The arrows indicate corresponding positions in successive micrographn. (a) First high-resolution scan; (b) second scan, after 8.6 s; (c) third scan, after approx. 90 s; (d) fourth scan, after 98 s; (e) fifth scan, after 210 s; (f) sixth scan, after 218 s. Scale, 3.4 A/mm.
depends on the exact distance from atom to electron beam. A variation in intensity exists from one osmium atom to another in the same image and in the image of a single atom from one scan to the next. The major consequence of the intensity variation is that it prevents a quantitative statement of the number of atoms contained in the spot. Except when a spot is both unusually bright and elongated, it has
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been assigned to a single osmium atom. This method probably leads to an underestimate of the number of unresolved osmium atom pairs. Because of the considerable irregularity of the support film thickness and the irregular intensity of the diffuse portion of the strand image, it is easier to identify small spots likely to be single osmium atoms after the image has been filtered to remove low spatial frequencies. Filtered images are shown in Figure I(c) and (d) and Figure 3(a) and (b). Figure 4 shows a portion of a labeled polycytidylic acid strand during six successive scans taken over a period of four minutes.
4. Discussion If electron microscopy of labeled systems is to establish macromolecular architecture, an exact relation between the observed positions of the osmium atoms and the chemical structure must exist. There are two major sources of ambiguity in this relation. First, because the polynucleotide molecule is very flexible, there exists a large number of configurations which it may assume on the support films. Except for the osmium atoms, the molecule is only faintly visible and its configuration cannot be determined from the micrograph. Even if the sugar-phosphate backbone is fully extended? the bases and attached stain atoms are free to rotate as they attach to the support film. A second ambiguity arises from the radiation damage to the molecule during imaging. The dose required to obtain clear atomic images is several orders of magnitude higher than that required to break the chemical bonds (Isaacson, 1976). If the stain atoms move appreciably before imaging occurs, information concerning the nucleic acid sequence will be lost. The average base-to-base spacing in these osmium-labeled polynucleotides is difficult to determine. As a test molecule, we have reacted single-stranded circular bacteriophage $X174 DNA with both osmium/bipyridine and osmium/dicarboxybipyridine. In either case, only cytosine and thymine would be expected to react with the osmium, giving 50% of the bases labeled (Chang et al., 1977). The lengths of osmium-labeled, intact circular molecules are presented in the histograms in Figure 5(a) and (b). Since +X174 has been shown to have 5375 bases (Sanger et al., 1977), the average base-to-base spacing can be determined by dividing the total length by 5375. Using bipyridine as the ligand yields an average spacing of 3.8 A f O-3 A. With the charged ligand dicarboxybipyridine, the average base-to-base spacing is 4.2 A f 0.3 A, or 10% greater. Both of these values are greater than unreacted #X174 (3 A) spread under the same conditions (data not shown), indicating that the addition of the osmium increases the spacing between bases. Since the sequence of $X174 includes all combinations of Py-Py, Pu-Py, etc., it is impossible to determine from this experiment the effect that the osmium would have on adjacent labeled nucleotides, as in our polyuridylic acid. It is likely that when every base in a particular region is labeled, the spacing is significantly greater than the 4 A observed above. Using the negatively charged ligand dicarboxybipyridine, we have successfully spread osmium-labeled 4X174 DNA t o an average base-to-base spacing of 6.4 A (data not shown). The osmium reaction causes very little damage to the nucleotide chain, since +X174 survives the osmium/bipyridine addition with very little breakage of the single-stranded circles. While there is some breakage with dicarboxybipyridine, at
OSMIUM-
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-(b) E IOL.2 -
i
0
0
Bx 6420
I o-5
I I.0
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Length (pm) I’IG. 5. Distribution of the length of 95x174 DNA molecules after reaction carboxybipyridine and (b) after reaction with osmium/bipyridina.
with (a) onmium/di-
least 20%, of the molecules remain intact. When spread for microscopy, the molecul~~s are extended, ea,sily measurable strands of uniform thickness. All of the osmiumlabeled polynucleotides are thinner and less rigid than unlabeled double-stranded DNA. The distribution of osmium atoms has been analysed in many micrographs of poly(U) and poly[d(A-T)]. Only occasionally do we observe a regularly spaced array of atoms as shown near the middle of Figure l(a) and (c). To measure the distribution of atoms, a hypothetical backbone was drawn along the strand and the distance from one atom to the next along this backbone was determined (Fig. 6). Histograms of the spacing between adjacent atoms for the micrographs of poly( U) a,nd poly[d(A-T)] are shown in Figure 7. In both cases there is a broad distribut,ion of spacings with a maximum at about 5 to 7 a for poly(U) and 9 to 10 .& for poly[d(A-T)]. The mean atom spacing for this strand of poly(U) is 6.7 A, with an average over many samples of about 6 d. For the poly[d(A-T)], which should have one osmium atom on every other base, the mean spacing is 10.4 A. Since the base-to-has;ch spacing is at least 4 8, and probably much more in poly(U), these measurements compare favorably with the expected values. Therefore it seems that most, if not) all, of the osmium atom markers can be identified in our micrographs. Nuclear magnetic resonance studies of osmium/bipyridine-labeled dinucleotides (ApU) by Daniel & Behrman (1976) suggest that there are stacking interactions between the bipyridine moiety and adjacent bases. It seemed possible that these interactions could cause gross distortion of the molecules and lead to unrecognizable patterns of atoms in the micrographs. This prompted the introduction of the negaDivcly charged analog dicarboxybipyridine, which was expect’ed to prevent any
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FIG. 6. (a) Micrograph of poly(U) stained with osmium/bipyridine; (b) same micrograph after computer filtering to remove low-frequency variation in the image; (c) interpretation of the miorograph indicating the positions of the atoms and a hypothetical backbone. S indicates the spacing between 2 adjacent atomdmeasured along the backbone. The bar represents 10 A.
0
3
6
9
12
15
18
21 -
spmg(Bi Pm. 7. Distribution of atom spacing along the strand. (a) Measurements with osmium/bipyridine (Fig. l(a)). (b) M easurements from poly[d(A-T)] dicarboxybipyridine (Fig. 2(a)).
from poly(U) stained stained with osmium/
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stacking interactions between adjacent labeled nucleotides. The strands in this case were somewhat straighter and more uniform in width than with the uncharged ligand, but showed no additional regularity in atom spacing. In order to assay any movement of the osmium atoms during imaging. two or more successive scans’ were recorded. The first scan was preceded by only a feu low-magnification scans to locate molecules of grossly appropriate configuration. Generally, this low-magnification scan results in less than 10% of the dose during a, high-magnification scan. Osmium atoms were identified on each scan and relative movement was noted by overlaying the images. Figure 8 shows histograms of t,hr includes atoms atom movement from Figures 1 and 2. The entry “not assignable” which were located in only one of the micrographs. Some of the assignments are uncerta,in and depend on the judgment of the person performing the comparison. However, the histograms indicate generally that 60% of the osmium atoms move less than 5 A from one scan to the next. Figure 4 represents an attempt to minimize the movement of the osmium atoms. This micrograph was obtained using an electron dose of less than one-third the usual dose. Furthermore, after the strands had been deposited on a 30 A carbon film, another layer of carbon about 100 A thick was indirectly evaporated over the top. The lower beam dosage gives a statistically poorer micrograph, while the thicker carbon film yields a higher background. Both conditions lead to more ambiguity in identifying individual osmium atoms. The amount of atom movement het’ween any two successive scans or over a period of several minutes was the same for this specimen as that, observed with uncoated specimens at higher beam dosages. The measurement of atomic movement from the first scan to the second does not indicate how much movement may have occurred before the recording of the first scan. It is possible that a gross rearrangement may have occurred during the lowmagnification scan or during the first few electrons of each high-resolution picture element. If the sample consisted of randomly distributed atoms, there would be no way to deal with this possibility. However, since the osmium atoms are initially attached to the polynucleotides, they are constrained to lie within a narrow band about the strand. From the expected bond lengths, the poly(U) specimen should he
I
I
I
~~~~~r mgnoble
Relotlw
(a)
movement
assignable
(5)
ibi
FIG. 8. Histogram of the measured movement of osmium atoms between successive scrtns 8.5 s apart. (a) Measurements from poly(U) stained with osmium/bipyridine (Fig. 1). (b) Measurements from poly[d(A-T)] stained with osmium/dicarboxybipyidine (Figs 2 end 3).
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18 A wide, which is approximately the width of the band actually measured in the micrographs. The dose delivered to the molecule during the low-magnification scan is probably sufficient to disrupt the osmium-nucleotide bonds (Isaacson, 1976). In general, several minutes elapse between the low and high-magnification scans. If the osmium atoms were free to diffuse in all directions during this time, one would not expect to find the well-defined band. If the movement observed between the first and second highmagnification scans is treated as a random diffusion process, it implies that the band containing the osmium atoms would widen to about 50 A by the sixth scan. The width of the band does not widen and remains between 17 and 20 A. Therefore it seems that the atoms are not randomly diffusing. This result is obtained for samples which have received a second coat of carbon and those which are uncoated. A possible explanation of the non-random nature of the osmium atom movement is suggested by our finding during microscopy with thin substrates of graphite crystals (White et al., 1971). It has been observed that molecules deposited on the thin graphite substrates almost always adhere to the boundary between two crystallites. (Unpublished observation.) If the carbon support film used here is considered to be a mosaic of very small graphite crystals, one might expect the osmium atoms to adhere to the crystal boundaries after being freed from their initial molecular site. The positions and movement of the osmium atoms at the 3 to 5 A level may be influenced by these interactions. Isaacson et al. (1976) measured the jump frequency and two-dimensional diffusion constant for atoms of uranium adsorbed to thin carbon films. The values given there for jump frequency, Ye = (5&2) x 1O-4 per second, and diffusion constant D = 4 x 10m3 A2 per second, are considerably lower than the same measures of atom motion calculated from the data shown here. We obtain vT = (6+2) x 10e2 per second and D = (4*2) >( 10-l A2 per second as average values from the data in Figure 1. The errors quoted take into account statistically probable errors and the variation likely due to individual differences in identifying atoms. Movements less than 3 A have been ignored in calculating the values. These measures would be expected to be independent of the time interval used in the calculation only if the atom movement is random. Motion which is correlated among atoms or where the total distance an atom may move from its initial position is limited are examples in which the measures of motion given above would not be time independent. Isaacson et al. (1976) use an interval of 100 seconds, while we used 8.5 seconds as the interval between scans. Some of the difference in our measurements may be due to the different time interval used. The path of several individual atoms has been traced by Isaacson et al. (1977). The paths seem to consist of many small jumps and a few much larger ones, suggesting non-random movement. The fact that the width of the band in which most of the osmium atoms initially attached to the polynucleotide are found does not increase over a period of several minutes is certainly evidence contradicting random motion. The evidence presented by Isaacson et al. (1976) that the movement of uranium atoms adsorbed on a carbon film is due to thermal energy, and the experiments of Dubochet (1975) and Ramamurti et al. (1975) indicating reduced specimen mass loss at low temperature suggest that there would be reduced movement of the labeling atoms if the specimen were observed at low temperature. Wall et al. (1977) have observed that there is a decrease in the amount of uranium atom movement seen when
OSMIUM-
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lowering the sample temperature from -40°C to -lOO”C, but the change is small and the jump frequency they obtain is much nearer what we obtain here at room temperature, Whether still lower temperatures will reduce the motion significantly cannot be predicted. The results presented in this paper differ from those obtained by Langmore & Crewe (1974) with mercury-stained nucleic acids. Their results indicated that the mercury remained on or near the strand only for electron doses far below that required for single atom imaging. The possibility that osmium would be a more stable atomic species was suggested by Langmore (1975). Whiting & Ottensmeyer (1972) have also obtained images of osmium-labeled polynucleotides. However, because of the different, mode of microscopy, they are unable to obtain successive exposures of the same area and therefore present no data on atom movement.
5. Conclusions The micrographs presented here show that it is possible to obtain images of single osmium atoms associated by specific chemical bonds with polynucleotides. Although the bonds are broken during the imaging, the osmium atoms are found to be contained within the narrow band of the polynucleotide strand and to move less than 5 a between scans. These results suggest that it might be possible to obta.in structural information on macromolecules to a resolution of perhaps even 5 d using osmium as a specific stain. Other heavy metals such as platinum (Germinario, Beer $ Wiggins, unpublished work) and uranium can probably also be used. With this assurance a wide variety of biological problems can now be attacked using the many known organo-metallic reactions to produce specific staining. Undoubtedly, still other specific staining reactions can be developed with the prospect of molecular microscopy providing the incentive. Indeed, we are continuing efforts along these lines in our laboratory. However, the kind of atom movements seen in the specimens studied here represents a major obstacle to using heavy-atom labels to determine nucleic acid base sequence. The work reported here dealt with stained polynucleotides. It cannot be inferred that the same amount of movement would occur if the heavy atoms were coupled to another macromolecule, for example a protein.
The authors gratefully acknowledge many valuable conversations with Dr Set11 Rose. The spatial filtering program was written by Ishik C. Tuna. This work was supported by National Institutes of Health grant no. GM08968.
REFERENCES Case, F. H. (1946). J. Amer. Chem. Sot. 68, 2574-2577. Chang, C. H., Beer, M. & Marzilli, L. G. (1977). Biochemislry, 16, 33-.38. Cole, M. D., Rose, S. D., Wiggins, J. W. & Beer, M. (1976). In Proc. 34th Ann. EMSA &feeting, (Bailey, G. W., ed.), pp. 328-329, Claitor’s, Baton Rouge, Louisiana. C’rcxwe, A. V., Langmore, J. P. & Isaacson, M. D. (1975). In Physical Aspects of Electron Microscopy and Microbeam Analysis (Siegel, B. M. & Beaman, D. K., eds), pp. 47-62, John Wiley, New York. Daniel, F. B. & Behrman, E. J. (1976). Biochemistry, 15, 565-568. Dubochet, J. (1975). J. Ultrastruct. Res. 52, 276-288.
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Engel, A., Wiggins, J. W. & Woodruff, D. C. (1974). J. AppZ. Phys. 45, 2739-2747. Formanek, H., Muller, M., Hahn, M. H. & Koller, Th. (1971). NatzLrwissenschaften, 58, 339-344. Hart, R. K., Kassner, T. F. & Marin, J. K. (1970). Phil. Mag. 21, 453-467. Hashimoto, H., Kumao, A., Hino, K., Yatsumato, Y. & Ono, A. (1971). Jup. J. Appl. Phya. 10, 1115-1116. Isaacson, M. S. (1976). In Principles and Techniques of Electron Microscopy: Biological Applicationns (Hayat, M. A., ed.), vol. 7, pp. l-78, Van Nostrand Reinhold, New York. Isaacson, M., Langmore, J. & Wall, J. (1974). In Proc. 7th Ann. SEM Symp. (Johari, 0. & Corvin, I., eds), pp. 19-26, IIT Research Institute, Chicago. Issacson, M. S., Langmore, J., Parker, N. W., Kopf, D. & Utlaut, M (1976). Ultramicroscopy, 1, 359-376. Isaacson, M. S., Kopf, D., Utlaut,, M., Parker, N. W. & Crewe, A. V. (1977). Proc. Nat. Acud. Sci., U.S.A. 74, 1802-1806. Langmore, J. (1975). Ph.D. Thesis, University of Chicago. Langmore, J. & Crewe, A. V. (1974). In Proc. 32nd Ann. EMSA Meeting, (Arceneux, C. J., ed), pp. 376--377, Claitor Publishing, Baton Rouge, Louisiana. 1, 156-158. Ramamurti, K., Crewe, A. V. & Isaacson, M. S. (1975). Ultramicroscopy, Sanger, F., Air, G. M., Barrell, B. G., Brown, N. L., Coulson, A. R., Fiddes, J. C., Hutchison C. A., III, Slocombe, P. M. & Smith, M. (1977). Nature (London), 265, 687-695. Vollenwieder, H. J., Sogo, J. M. & Koller, Th. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 83-87. Wall, J., Langmore, J ., Isaacson, M. & Crewe, A. V. (1974). Proc. IVat. Acud. Sci.,U.S.A. 71, l-5. Wall, J. S., Hainfeld, J. F. & Bittner, J. W. (1977). Ultramicroscopy, in the press. White, J. R., Beer, M. &Wiggins, J. W. (1971). Micron, 2, 412-427. Whiting, R. F. & Ottensmeyer, F. P. (1972). J. Mol. Biol. 67, 173-181. Wiggins, J. W., Beer, M., Woodruff, D. C. & Zubin, J. (1974). In Proc. 32nd Ann. EMSA Meeting, (Arceneux, C. J. ed). pp. 404-405, Claitor’s Publishing, Baton Rouge. Woodruff, D., Wiggins, J. W. & Zubin, J. (1974). In Proc. 32nd Ann. EMSA Meeting, (Arceneux, C. J., ed), pp. 430-431, Claitor’s Publishing, Baton Rouge. Zeitler, E. & Thomson, M. G. R. (1970). Optik, 31, 281-291, 359-366.
(1977) 117, 387400
Molecular Microscopy of Labeled Polynucleotides : Stability of Osmium Atoms M. D. COLE, J. W. WIGGINS
AND M. BEER
The T. C. Jenkins Departwhent of Biophysics The Johns Hopkins University Baltimore, Md 21218, U.S.A. (Received 20 May 1977, and in revised forwa 25 August
1977)
Polynucleotides and modified polynucleotides reacted with osmium tetroxide and bipyridine or dicarboxybipyridine to bind one or two osmium atoms per nucleotide were viewed in a high-resolution scanning transmission electron microscope under conditions where the osmium atoms are visible. The micrographs show the individual osmium atoms in a configuration determined by their specific chemical bonding with the nucleic acid molecule and with a number and average spacing that is consistent with the expected value. Although the bonds are broken during the imaging, many osmium atoms are found to be contained within the narrow band of the polynucleotide strand and to move less than 5 A between scans of the same area. These results suggest that it is possible to obtain structural information on macromolecules to a resolution of 5 to 10 A using osmium as a specific stain.
1. Introduction Many of the central problems in modern biology can be attacked powerfully if single sites in macromolecules or macromolecular heavy atoms are bound to particular assemblies, and the sites so labeled are recognized in the electron microscope. For example, such specific labeling might be used to localize nucleic acid strands or individual protein molecules in ribosomes, nucleosomes or other nucleoprotein complexes. Considerable chemical information is now available which makes possible the preparation of appropriately labeled macromolecular systems for electron microscopy. The visualization of heavy atoms has now been realized by several laboratories using either the particularly suitable high-resolution scanning transmission electron microscope (Wall et al., 1974; Coleet al., 1976) or conventional electron microscopes (Whiting & Ottensmeyer, 1972 ; Hashimoto et al., 1971; Formanek et al., 1971). The stability of heavy atoms incorporated into macromolecules during the preparation of specimens and during electron microscopy are key questions which have not been adequately answered. Indeed, some notable failures in obtaining structural insight from micrographs of labeled macromolecular systems have cast doubt on the feasibility of the approach (Langmore & Crewe, 1974). In this paper we demonstrate that heavy atoms bound to biological macromolecules are detectable; that over long stretches their number and distribution are consistent with expectation, and that pairs of successive micrographs indicate an average movement of about 3 a for each heavy atom from the first to second micrograph, neglecting those that ha,ve moved so far that they cannot be identified. 3; 387
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2. Materials and Methods Heavy-atom-labeled specimens were prepared as described by Chang et al. (1977) by treating polynucleotides with 5 mM-osmium tetroxide in 5 m&f-bipyridine or 4,4’-dicarboxy2,2’-bipyridine for 24 h at room temperature followed by gel filtration chromatography on Sephadex G50. Polyuridylic acid acquires 1 osmium atom/nucleotide under these conditions. Each thymidine in poly[d(A-T)] will also acquire 1 osmium atom (Chang et aZ., 1977). To obtain a sample containing 2 heavy atoms/nucleotide, polycytidylic acid was allowed to react with o-furfuryl-hydroxylamine in the presence of sodium bisulfite prior to treatment with osmium tetroxide. Under these conditions the modified polymer takes up 2 heavy atoms/nucleotide (Rose & Beer, unpublished work). Specimens prepared in the manner described above contain covalently bound, heavy-atom labels at essentially every nucleotide. 4,4’-Dicarboxy-2,2’-bipyridine was synthesized as described by Case (1946). Thin carbon support films were prepared by indirect evaporation from a resistanceheated rod onto freshly cleaved mica. The thinnest, smoothest films were obtained by relatively slow evaporation. Thickness was assayed in the scanning transmission electron microscope by the electron scattering intensity. A typical film was 30 A thick. The thin films were supported on carbon-coated fenestrated plastic films. A number of bright spots, assumed to be heavy element atoms, are seen in the background of the images. The density of background atoms depends on the preparation techniques and ranges between 2 and 3 atoms/lo3 A”. In an attempt to minimize the number of these spots, spectroscopically pure carbon (National AGKSP) and deionized water (from a Millipore Milli-Q system; resistivity = 18 Mohm cm-‘) were used (Isaacson et al., 1974). Grid materials other than copper, a variety of plastic materials for fenestrated films, and treating the films with chelating agents were tried with no consistent improvement over the level seen in the images presented here. The polynucleotide strands were freed from virtually all contaminating salts by gel filtration chromatography using deionized water as the eluant. This solution was then made 1O-30/o (w/v) benzyldimethylalkylammonium chloride and spread as a monolayer on deionized water (Vollenweider et al., 1975). The polynucleotide preparations were viewed in a high-resolution scanning transmission electron microscope constructed at Johns Hopkins (Wiggins et al., 1974). The beam energy was between 47 and 50 keV in all instances. Total pressure throughout the microscope was below 10mQ Torr. Data were recorded from two electron detectors. Each detector consists of a CaF(Eu) scintillator optically coupled to a photomultiplier. One, designated the central detector, collected the electrons falling within the cone of illumination with a half angle of 13 mrad. The other detector was an annular disc of scintillator collecting the electrons between 13 mrad and approximately 156 mrad. The images shown here consist of the ratio of the annular detector signal to the central detector signal; therefore, they are dark field images. The detector signals and the ratio were recorded by a digital scanning system (Woodruff et al., 1974). The time for which the beam samples each point on the specimen is 30 ps. The electron dosage was determined by two separate methods. One method was a calculation from the known total field emission current, demagnification, and aperture angle. The other method was through calibration of the scintillator-photomultiplier sensitivity. The electron dose in all cases was between lo3 and lo4 e/AZ. The specimen was first searched at low magnification to locate polynucleotide strands. This search resulted in an irradiation of the strand of no more than 10% of the dose in a single high magnification scan. Focusing was accomplished on an adjacent area and then the scan was shifted electrically to the strand where successive scans at high magnification were recorded. The final images were produced by a digital film scanner/writer. Although the ultra-high vacuum environment of the microscope contributes no matter to the sample, contamination can be observed due to surface diffusion of matter (Hart et al., 1970). To avoid contamination, each sample was irradiated by an infra-red lamp while in the airlock (Isaacson et al., 1974). It was then inserted into the high vacuum chamber while still warm. After this procedure there was no detectable contamination. Spatial filtering of the images was accomplished digitally with the PDP 1 l/20 processor,
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which is an integral part of our system. The convolution integral J(Z) = j J(C) *A(.z - [) di was evaluated where I(Z) is the micrograph intensity and A(z) is an envelope function defining the extent of spatial filtering. This integral is identical to .9-1[~(1(z))*9(A(z))], which is the usual form of a spatially filtered function. The A(z) value used W&Ban integer approximation to a 2-dimensional Gaussian function centered on the pixel being evaluated, and including 32, 64 or 128 pixels total. image. The spatial frequencies are attenuated above 0.1 A-l.J(z) is a low frequency The high frequency image K(X) is obtained aa K(z) = I(Z) - J(a) + C, where C, a constant, is chosen large enough that K(z) is always positive. A more conventional Fourier transform and filtering program, or use of a larger memory and more time to perform the convolution described here, would allow a wider choice of the envelope function A(z). The speed of this short filtering r&tine and its availability on the on-line computer make it worthwhile. Comparison of the filtered and unfiltered images presented here and filtering of known test patterns give confidence that no unexpected feature is being introduced into the filtered images. The criteria used to assign a spot aa an osmium atom were that it be more intense than its surrounding in the unfiltered image, that it be more intense than the general background in the filtered image, and that it have a size comparable to the resolution of the microscope. Those spots larger than the microscope resolution and of sufficient intensity M’ere assigned as 2 osmium atoms. Although the assignment technique is quite subjective, ahnost any observer will agree on the quantitative meas&es we have derived from the micrographs within $200/& Figure 6 shows an example of how we have interpreted a port.ion of Fig. l(a).
3. Results Figures 1, 2, 3 and 4 show examples of polynucleotide strands that have been labeled with osmium. The osmium atoms appear as small white spots. In addition, the organic portion of the strand, and probably additional adsorbed organic material, produce diffuse light areas surrounding the osmium atoms. These images show individual atoms in a configuration resulting from their specific chemical association with the nucleic acid molecules. Examination of unreacted polynucleotides shows strands that are only faintly visible, if at all, with no identifiable atoms over those expected from the random background. The intensity of the osmium atoms with respect to the support film, the lack of phase contrast effects in the support film, and the general appearance of the images are all in accord with scanning transmission electron microscope imaging theory (Zeitler 6 Thomson, 1970), calculations of single atom appearance (Engel et al., 1974), and previous scanning transmission microscopy of single atoms (Crewe et al., 1975). If the images are examined closely, linear striations in the direction of the scan lines are seen superimposed on the regular features. These striations indicate movement during the scanning time. The fact that the striations are in the scan direction does not necessarily indicate that the actual movement was in that direction. Movement in any direction in the specimen plane, of period comparable to the time the beam samples each specimen point, will show up as though in the scan direction. The movement may be due to two sources. One possibility is that the components of the specimen move during electron exposure. This seems to be true and is discussed below. Another less fundamental source of movement is from the microscope itself. Instabilities, both mechanical and electrical, may cause abrupt translation of the heam with respect to the specimen. We are currently unable to separate the contribution due to specimen movement from the instrumental instability. The same movements of beam relative to specimen might give rise to a variation in the
intensity
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Fm. 1. Polyuridylic acid labeled with 1 osmium atom/nucleotide using osmium/bipyridino. (a) First high-resolution scan. (b) Second scan taken 8.6 s later. (0) First scan after filtering with the computer to remove low frequency variations in the micrograph. (d) Second scan after filtering. The horizontal width of each micrograph is 410 A.
OSMIUM-
Poly[d( A-T)] labeled ‘(a) First high- resolution
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with 1 osmium atom/thymidine using osmium/die acan. (b) Second scan. The horizontal width of each IT
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Fro. 3. Images from Figure
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(a) First scan. (b) Second scan.
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FIG. 4. Polycytidylic acid labeled with 2 osmium etoms/nucleotide (see Materials and Methods). The specimen was prepared by adsorbing the osmium-labeled polynucleotides to a thin 30 A carbon film and then evaporating a 100 A layer of carbon over the strands. All images have been low-frequency filtered. The arrows indicate corresponding positions in successive micrographn. (a) First high-resolution scan; (b) second scan, after 8.6 s; (c) third scan, after approx. 90 s; (d) fourth scan, after 98 s; (e) fifth scan, after 210 s; (f) sixth scan, after 218 s. Scale, 3.4 A/mm.
depends on the exact distance from atom to electron beam. A variation in intensity exists from one osmium atom to another in the same image and in the image of a single atom from one scan to the next. The major consequence of the intensity variation is that it prevents a quantitative statement of the number of atoms contained in the spot. Except when a spot is both unusually bright and elongated, it has
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been assigned to a single osmium atom. This method probably leads to an underestimate of the number of unresolved osmium atom pairs. Because of the considerable irregularity of the support film thickness and the irregular intensity of the diffuse portion of the strand image, it is easier to identify small spots likely to be single osmium atoms after the image has been filtered to remove low spatial frequencies. Filtered images are shown in Figure I(c) and (d) and Figure 3(a) and (b). Figure 4 shows a portion of a labeled polycytidylic acid strand during six successive scans taken over a period of four minutes.
4. Discussion If electron microscopy of labeled systems is to establish macromolecular architecture, an exact relation between the observed positions of the osmium atoms and the chemical structure must exist. There are two major sources of ambiguity in this relation. First, because the polynucleotide molecule is very flexible, there exists a large number of configurations which it may assume on the support films. Except for the osmium atoms, the molecule is only faintly visible and its configuration cannot be determined from the micrograph. Even if the sugar-phosphate backbone is fully extended? the bases and attached stain atoms are free to rotate as they attach to the support film. A second ambiguity arises from the radiation damage to the molecule during imaging. The dose required to obtain clear atomic images is several orders of magnitude higher than that required to break the chemical bonds (Isaacson, 1976). If the stain atoms move appreciably before imaging occurs, information concerning the nucleic acid sequence will be lost. The average base-to-base spacing in these osmium-labeled polynucleotides is difficult to determine. As a test molecule, we have reacted single-stranded circular bacteriophage $X174 DNA with both osmium/bipyridine and osmium/dicarboxybipyridine. In either case, only cytosine and thymine would be expected to react with the osmium, giving 50% of the bases labeled (Chang et al., 1977). The lengths of osmium-labeled, intact circular molecules are presented in the histograms in Figure 5(a) and (b). Since +X174 has been shown to have 5375 bases (Sanger et al., 1977), the average base-to-base spacing can be determined by dividing the total length by 5375. Using bipyridine as the ligand yields an average spacing of 3.8 A f O-3 A. With the charged ligand dicarboxybipyridine, the average base-to-base spacing is 4.2 A f 0.3 A, or 10% greater. Both of these values are greater than unreacted #X174 (3 A) spread under the same conditions (data not shown), indicating that the addition of the osmium increases the spacing between bases. Since the sequence of $X174 includes all combinations of Py-Py, Pu-Py, etc., it is impossible to determine from this experiment the effect that the osmium would have on adjacent labeled nucleotides, as in our polyuridylic acid. It is likely that when every base in a particular region is labeled, the spacing is significantly greater than the 4 A observed above. Using the negatively charged ligand dicarboxybipyridine, we have successfully spread osmium-labeled 4X174 DNA t o an average base-to-base spacing of 6.4 A (data not shown). The osmium reaction causes very little damage to the nucleotide chain, since +X174 survives the osmium/bipyridine addition with very little breakage of the single-stranded circles. While there is some breakage with dicarboxybipyridine, at
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Length (pm) I’IG. 5. Distribution of the length of 95x174 DNA molecules after reaction carboxybipyridine and (b) after reaction with osmium/bipyridina.
with (a) onmium/di-
least 20%, of the molecules remain intact. When spread for microscopy, the molecul~~s are extended, ea,sily measurable strands of uniform thickness. All of the osmiumlabeled polynucleotides are thinner and less rigid than unlabeled double-stranded DNA. The distribution of osmium atoms has been analysed in many micrographs of poly(U) and poly[d(A-T)]. Only occasionally do we observe a regularly spaced array of atoms as shown near the middle of Figure l(a) and (c). To measure the distribution of atoms, a hypothetical backbone was drawn along the strand and the distance from one atom to the next along this backbone was determined (Fig. 6). Histograms of the spacing between adjacent atoms for the micrographs of poly( U) a,nd poly[d(A-T)] are shown in Figure 7. In both cases there is a broad distribut,ion of spacings with a maximum at about 5 to 7 a for poly(U) and 9 to 10 .& for poly[d(A-T)]. The mean atom spacing for this strand of poly(U) is 6.7 A, with an average over many samples of about 6 d. For the poly[d(A-T)], which should have one osmium atom on every other base, the mean spacing is 10.4 A. Since the base-to-has;ch spacing is at least 4 8, and probably much more in poly(U), these measurements compare favorably with the expected values. Therefore it seems that most, if not) all, of the osmium atom markers can be identified in our micrographs. Nuclear magnetic resonance studies of osmium/bipyridine-labeled dinucleotides (ApU) by Daniel & Behrman (1976) suggest that there are stacking interactions between the bipyridine moiety and adjacent bases. It seemed possible that these interactions could cause gross distortion of the molecules and lead to unrecognizable patterns of atoms in the micrographs. This prompted the introduction of the negaDivcly charged analog dicarboxybipyridine, which was expect’ed to prevent any
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FIG. 6. (a) Micrograph of poly(U) stained with osmium/bipyridine; (b) same micrograph after computer filtering to remove low-frequency variation in the image; (c) interpretation of the miorograph indicating the positions of the atoms and a hypothetical backbone. S indicates the spacing between 2 adjacent atomdmeasured along the backbone. The bar represents 10 A.
0
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spmg(Bi Pm. 7. Distribution of atom spacing along the strand. (a) Measurements with osmium/bipyridine (Fig. l(a)). (b) M easurements from poly[d(A-T)] dicarboxybipyridine (Fig. 2(a)).
from poly(U) stained stained with osmium/
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stacking interactions between adjacent labeled nucleotides. The strands in this case were somewhat straighter and more uniform in width than with the uncharged ligand, but showed no additional regularity in atom spacing. In order to assay any movement of the osmium atoms during imaging. two or more successive scans’ were recorded. The first scan was preceded by only a feu low-magnification scans to locate molecules of grossly appropriate configuration. Generally, this low-magnification scan results in less than 10% of the dose during a, high-magnification scan. Osmium atoms were identified on each scan and relative movement was noted by overlaying the images. Figure 8 shows histograms of t,hr includes atoms atom movement from Figures 1 and 2. The entry “not assignable” which were located in only one of the micrographs. Some of the assignments are uncerta,in and depend on the judgment of the person performing the comparison. However, the histograms indicate generally that 60% of the osmium atoms move less than 5 A from one scan to the next. Figure 4 represents an attempt to minimize the movement of the osmium atoms. This micrograph was obtained using an electron dose of less than one-third the usual dose. Furthermore, after the strands had been deposited on a 30 A carbon film, another layer of carbon about 100 A thick was indirectly evaporated over the top. The lower beam dosage gives a statistically poorer micrograph, while the thicker carbon film yields a higher background. Both conditions lead to more ambiguity in identifying individual osmium atoms. The amount of atom movement het’ween any two successive scans or over a period of several minutes was the same for this specimen as that, observed with uncoated specimens at higher beam dosages. The measurement of atomic movement from the first scan to the second does not indicate how much movement may have occurred before the recording of the first scan. It is possible that a gross rearrangement may have occurred during the lowmagnification scan or during the first few electrons of each high-resolution picture element. If the sample consisted of randomly distributed atoms, there would be no way to deal with this possibility. However, since the osmium atoms are initially attached to the polynucleotides, they are constrained to lie within a narrow band about the strand. From the expected bond lengths, the poly(U) specimen should he
I
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FIG. 8. Histogram of the measured movement of osmium atoms between successive scrtns 8.5 s apart. (a) Measurements from poly(U) stained with osmium/bipyridine (Fig. 1). (b) Measurements from poly[d(A-T)] stained with osmium/dicarboxybipyidine (Figs 2 end 3).
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18 A wide, which is approximately the width of the band actually measured in the micrographs. The dose delivered to the molecule during the low-magnification scan is probably sufficient to disrupt the osmium-nucleotide bonds (Isaacson, 1976). In general, several minutes elapse between the low and high-magnification scans. If the osmium atoms were free to diffuse in all directions during this time, one would not expect to find the well-defined band. If the movement observed between the first and second highmagnification scans is treated as a random diffusion process, it implies that the band containing the osmium atoms would widen to about 50 A by the sixth scan. The width of the band does not widen and remains between 17 and 20 A. Therefore it seems that the atoms are not randomly diffusing. This result is obtained for samples which have received a second coat of carbon and those which are uncoated. A possible explanation of the non-random nature of the osmium atom movement is suggested by our finding during microscopy with thin substrates of graphite crystals (White et al., 1971). It has been observed that molecules deposited on the thin graphite substrates almost always adhere to the boundary between two crystallites. (Unpublished observation.) If the carbon support film used here is considered to be a mosaic of very small graphite crystals, one might expect the osmium atoms to adhere to the crystal boundaries after being freed from their initial molecular site. The positions and movement of the osmium atoms at the 3 to 5 A level may be influenced by these interactions. Isaacson et al. (1976) measured the jump frequency and two-dimensional diffusion constant for atoms of uranium adsorbed to thin carbon films. The values given there for jump frequency, Ye = (5&2) x 1O-4 per second, and diffusion constant D = 4 x 10m3 A2 per second, are considerably lower than the same measures of atom motion calculated from the data shown here. We obtain vT = (6+2) x 10e2 per second and D = (4*2) >( 10-l A2 per second as average values from the data in Figure 1. The errors quoted take into account statistically probable errors and the variation likely due to individual differences in identifying atoms. Movements less than 3 A have been ignored in calculating the values. These measures would be expected to be independent of the time interval used in the calculation only if the atom movement is random. Motion which is correlated among atoms or where the total distance an atom may move from its initial position is limited are examples in which the measures of motion given above would not be time independent. Isaacson et al. (1976) use an interval of 100 seconds, while we used 8.5 seconds as the interval between scans. Some of the difference in our measurements may be due to the different time interval used. The path of several individual atoms has been traced by Isaacson et al. (1977). The paths seem to consist of many small jumps and a few much larger ones, suggesting non-random movement. The fact that the width of the band in which most of the osmium atoms initially attached to the polynucleotide are found does not increase over a period of several minutes is certainly evidence contradicting random motion. The evidence presented by Isaacson et al. (1976) that the movement of uranium atoms adsorbed on a carbon film is due to thermal energy, and the experiments of Dubochet (1975) and Ramamurti et al. (1975) indicating reduced specimen mass loss at low temperature suggest that there would be reduced movement of the labeling atoms if the specimen were observed at low temperature. Wall et al. (1977) have observed that there is a decrease in the amount of uranium atom movement seen when
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lowering the sample temperature from -40°C to -lOO”C, but the change is small and the jump frequency they obtain is much nearer what we obtain here at room temperature, Whether still lower temperatures will reduce the motion significantly cannot be predicted. The results presented in this paper differ from those obtained by Langmore & Crewe (1974) with mercury-stained nucleic acids. Their results indicated that the mercury remained on or near the strand only for electron doses far below that required for single atom imaging. The possibility that osmium would be a more stable atomic species was suggested by Langmore (1975). Whiting & Ottensmeyer (1972) have also obtained images of osmium-labeled polynucleotides. However, because of the different, mode of microscopy, they are unable to obtain successive exposures of the same area and therefore present no data on atom movement.
5. Conclusions The micrographs presented here show that it is possible to obtain images of single osmium atoms associated by specific chemical bonds with polynucleotides. Although the bonds are broken during the imaging, the osmium atoms are found to be contained within the narrow band of the polynucleotide strand and to move less than 5 a between scans. These results suggest that it might be possible to obta.in structural information on macromolecules to a resolution of perhaps even 5 d using osmium as a specific stain. Other heavy metals such as platinum (Germinario, Beer $ Wiggins, unpublished work) and uranium can probably also be used. With this assurance a wide variety of biological problems can now be attacked using the many known organo-metallic reactions to produce specific staining. Undoubtedly, still other specific staining reactions can be developed with the prospect of molecular microscopy providing the incentive. Indeed, we are continuing efforts along these lines in our laboratory. However, the kind of atom movements seen in the specimens studied here represents a major obstacle to using heavy-atom labels to determine nucleic acid base sequence. The work reported here dealt with stained polynucleotides. It cannot be inferred that the same amount of movement would occur if the heavy atoms were coupled to another macromolecule, for example a protein.
The authors gratefully acknowledge many valuable conversations with Dr Set11 Rose. The spatial filtering program was written by Ishik C. Tuna. This work was supported by National Institutes of Health grant no. GM08968.
REFERENCES Case, F. H. (1946). J. Amer. Chem. Sot. 68, 2574-2577. Chang, C. H., Beer, M. & Marzilli, L. G. (1977). Biochemislry, 16, 33-.38. Cole, M. D., Rose, S. D., Wiggins, J. W. & Beer, M. (1976). In Proc. 34th Ann. EMSA &feeting, (Bailey, G. W., ed.), pp. 328-329, Claitor’s, Baton Rouge, Louisiana. C’rcxwe, A. V., Langmore, J. P. & Isaacson, M. D. (1975). In Physical Aspects of Electron Microscopy and Microbeam Analysis (Siegel, B. M. & Beaman, D. K., eds), pp. 47-62, John Wiley, New York. Daniel, F. B. & Behrman, E. J. (1976). Biochemistry, 15, 565-568. Dubochet, J. (1975). J. Ultrastruct. Res. 52, 276-288.
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Engel, A., Wiggins, J. W. & Woodruff, D. C. (1974). J. AppZ. Phys. 45, 2739-2747. Formanek, H., Muller, M., Hahn, M. H. & Koller, Th. (1971). NatzLrwissenschaften, 58, 339-344. Hart, R. K., Kassner, T. F. & Marin, J. K. (1970). Phil. Mag. 21, 453-467. Hashimoto, H., Kumao, A., Hino, K., Yatsumato, Y. & Ono, A. (1971). Jup. J. Appl. Phya. 10, 1115-1116. Isaacson, M. S. (1976). In Principles and Techniques of Electron Microscopy: Biological Applicationns (Hayat, M. A., ed.), vol. 7, pp. l-78, Van Nostrand Reinhold, New York. Isaacson, M., Langmore, J. & Wall, J. (1974). In Proc. 7th Ann. SEM Symp. (Johari, 0. & Corvin, I., eds), pp. 19-26, IIT Research Institute, Chicago. Issacson, M. S., Langmore, J., Parker, N. W., Kopf, D. & Utlaut, M (1976). Ultramicroscopy, 1, 359-376. Isaacson, M. S., Kopf, D., Utlaut,, M., Parker, N. W. & Crewe, A. V. (1977). Proc. Nat. Acud. Sci., U.S.A. 74, 1802-1806. Langmore, J. (1975). Ph.D. Thesis, University of Chicago. Langmore, J. & Crewe, A. V. (1974). In Proc. 32nd Ann. EMSA Meeting, (Arceneux, C. J., ed), pp. 376--377, Claitor Publishing, Baton Rouge, Louisiana. 1, 156-158. Ramamurti, K., Crewe, A. V. & Isaacson, M. S. (1975). Ultramicroscopy, Sanger, F., Air, G. M., Barrell, B. G., Brown, N. L., Coulson, A. R., Fiddes, J. C., Hutchison C. A., III, Slocombe, P. M. & Smith, M. (1977). Nature (London), 265, 687-695. Vollenwieder, H. J., Sogo, J. M. & Koller, Th. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 83-87. Wall, J., Langmore, J ., Isaacson, M. & Crewe, A. V. (1974). Proc. IVat. Acud. Sci.,U.S.A. 71, l-5. Wall, J. S., Hainfeld, J. F. & Bittner, J. W. (1977). Ultramicroscopy, in the press. White, J. R., Beer, M. &Wiggins, J. W. (1971). Micron, 2, 412-427. Whiting, R. F. & Ottensmeyer, F. P. (1972). J. Mol. Biol. 67, 173-181. Wiggins, J. W., Beer, M., Woodruff, D. C. & Zubin, J. (1974). In Proc. 32nd Ann. EMSA Meeting, (Arceneux, C. J. ed). pp. 404-405, Claitor’s Publishing, Baton Rouge. Woodruff, D., Wiggins, J. W. & Zubin, J. (1974). In Proc. 32nd Ann. EMSA Meeting, (Arceneux, C. J., ed), pp. 430-431, Claitor’s Publishing, Baton Rouge. Zeitler, E. & Thomson, M. G. R. (1970). Optik, 31, 281-291, 359-366.